tbuckley/cabot-search huggingface dataset:
Abstract 1224: UMIBB: A novel nonparametric Bayesian method improves robustness and sensitivity of analysis in pooled CRISPR-Cas9 screens leveraging unique molecular identifiers
Authors: Ashley Choi, Samuel Meier, Silvia Fenoglio, Tianshu Feng, Justin Engel, Binzhang Shen, Shangtao Liu, Teng Teng, Tenzing Khendu, Alan Huang, Jannik Andersen, Xuewen Pan, Yi Yu
Journal: Cancer Research (2022)

Abstract CRISPR-based functional genomics screens are a powerful tool for identifying synthetically lethal cancer drug targets. Current strategies for analyzing pooled CRISPR screens usually rely on signals from single guide RNAs (sgRNA) with differential relative abundance between two experimental conditions. However, conventional approaches are susceptible to false positives and false negatives driven by outlier cell clones, since the sgRNA abundance does not account for the heterogeneous phenotypes resulting from different editing outcomes of the same sgRNA. To overcome this, we added DNA barcodes to each sgRNA to create unique molecular identifiers (UMIs) for CRISPR libraries and developed a companion analytical platform that enables robust, industry-scale CRISPR screens. Here, we present UMIBB, a novel nonparametric Bayesian approach for analyzing UMI-CRISPR data. The number of UMIs with normalized count depletion or enrichment compared to the control experimental condition for each sgRNA is modeled by a beta-binomial distribution. The gene level statistics are derived by combining z-scores of the sgRNAs level posterior probabilities weighted by the number of UMIs in each sgRNA. This approach minimizes the impact of outlier cell clones on statistics and prioritizes genes with consistent count differentials across multiple UMIs in each gene. To assess the power of UMIBB, we benchmarked it on a low coverage (200X) genome-scale negative-selection screen, comparing with results from a high coverage (1000X) screen. These screens were conducted on KRAS mutant cancer celllines (A549) treated with trametinib or vehicle control. Despite the high noise level usually observed in lower coverage screens, our method was able to uncover most of the validated sensitizer genes for trametinib and achieved the highest sensitivity compared to conventional methods. Furthermore, we applied UMIBB on a genome-scale positive-selection screen and successfully identified novel genes (RAD18 and UBE2K) as key mediators of USP1 dependency in BRCA1 mutant cell lines. Our studies demonstrate that UMIBB is highly robust against false positives due to clonal heterogeneity and is more likely to identify true genetic interactions. Citation Format: Ashley Choi, Samuel Meier, Silvia Fenoglio, Tianshu Feng, Justin Engel, Binzhang Shen, Shangtao Liu, Teng Teng, Tenzing Khendu, Alan Huang, Jannik Andersen, Xuewen Pan, Yi Yu. UMIBB: A novel nonparametric Bayesian method improves robustness and sensitivity of analysis in pooled CRISPR-Cas9 screens leveraging unique molecular identifiers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1224.
Abstract 942: Development of customizable bladder cancer mouse model using CRISPR
Authors: Henkel L. Valentine, Uttam Satyal, Philip H. Abbosh
Journal: Cancer Research (2022)

Abstract Introduction: In forward genetics, a phenotype (sensitivity to therapy) is attributed to a genotype (mutation in relevant gene) based on findings in anecdotal cases (e.g., extreme responders) with little systematic/experimental study. Although powerful, this approach is not widely applicable due to the limited availability of clinical materials and throughput thus limiting the number and type of hypotheses that can be rigorously tested. However, a genotype-therapy sensitivity relationship could be measured in isogenic systems using reverse genetics, whereby mutations in relevant genes are introduced followed by evaluation of a phenotype in wild-type and mutant contexts. Since no such pre-clinical system exists for bladder cancer, we developed an immunocompetent mouse model to induce customizable isogenic tumor formation in bladder urothelium using CRISPR. Methods: Bladder urothelium was harvested from transgenic mice harboring LSL-Cas9/GFP in Rosa26 locus (as described by Platt et al) and grown as organoids. These organoids had a basal phenotype. AAV harboring Cre and sgRNA(s) were used to induce Cas9 and engineer mutations. Based on their frequent somatic alteration pathway in TCGA-BLCA, Tp53 and Rb1 were chosen for knockout first to establish ‘base organoids’. Additional genes related to PI3K/AKT/mTOR pathway were edited in base organoids to develop a series of organoids with different genotypes predicted to activate this signaling pathway. Sanger sequencing and western blot were used to confirm knockout. Kinase inhibitors targeting the mTOR pathway at different levels were assessed in organoids with different genotypes. The edited urothelial cells were grafted in mouse bladder to develop isogenic tumor to assess in vivo therapeutic vulnerability of in-vitro-prioritized kinase inhibitors matching the genotype of the allografted tumor. Results: Rb1, Tp53, and Pten were successfully knocked out in urothelial organoids. When treated with Ipataseritib, an AKT inhibitor, the triple knockout organoid was found to be 9X more sensitive than Trp53/Rb1 knockout organoids. In contrast, triple knockout organoids were 15X less sensitive to Gefitinib, an EGFR inhibitor, than Trp53/Rb1 knockout organoids, in line with published data. Edited organoids generate a neo-urothelium in murine bladder in short term assays. Conclusions: We developed a flexible bladder cancer mouse model to study mutation-therapy relationship using reverse genetics. This system is a plausible way to uncover unappreciated therapeutic vulnerabilities that probably exist in bladder cancer. Citation Format: Henkel L. Valentine, Uttam Satyal, Philip H. Abbosh. Development of customizable bladder cancer mouse model using CRISPR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 942.
Abstract 2275: Transcription factor landscape cataloguing highlights key insights in inflammatory breast cancer (IBC)
Authors: Diego A. Rosado-Tristani, Carlos S. Morales, Esther A. Peterson, Jose A. Rodríguez-Martínez
Journal: Cancer Research (2022)

Abstract Transcription factors are proteins that bind to DNA in a sequence-specific manner to regulate gene expression for normal cellular functions. Cancers have been shown to have aberrant transcriptional regulation. Therefore, identifying transcription factor landscapes will aid in characterizing complex diseases. As an example, breast cancer has many subtypes that are phenotypically and molecularly distinct. Inflammatory Breast Cancer (IBC) is rare, and the most aggressive form of breast cancer currently known. It is a poorly characterized subtype, and diagnosis often results in poor prognosis for patients. As such, we decided to explore the transcription factor landscape in IBC. This was accomplished employing the following strategy: 1) We assembled de novo transcriptomes for SUM149, an IBC cancer line and MCF7, a non-IBC cancer line using Trinity; 2) We translated the assembled transcriptomes using getORF from EMBOSS; 3) We identified putative transcription factors in the translated transcriptomes using CREPE. We then identified differentially expressed genes between SUM-149 and MCF-7 using DEseq2. Our results highlight differences in the transcription factor catalogues between IBC and non-IBC cancers. Of interest in SUM-149 is the gene BNC1, a member of the C2H2 zinc finger family of transcription factors. Dysregulation of this gene is associated with brain metastasis in breast cancers which is a common phenotype in IBC. BNC1-associated genes are linked to GO terms such as, epithelial cell differentiation, and regulation of alternative mRNA splicing, via spliceosome; however, BNC1 functions in IBC are yet to be determined. This analysis will bridge the gap in knowledge of key transcriptional regulators in IBC. Citation Format: Diego A. Rosado-Tristani, Carlos S. Morales, Esther A. Peterson, Jose A. Rodríguez-Martínez. Transcription factor landscape cataloguing highlights key insights in inflammatory breast cancer (IBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2275.
Abstract 5126: PIANOS: A platform independent and normalization free single-sample classifier for colorectal cancer
Authors: Du Cai, Haoning Qi, Wei Wang, Min-Er Zhong, Dejun Fan, Cheng-Hang Li, Xiaojian Wu, Feng Gao
Journal: Cancer Research (2022)

Abstract Objective: Identifying robust prognostic biomarkers of colorectal cancer (CRC) will significantly improve patients’ outcomes. However, due to the difference in detection methods, sequencing platforms and cohort populations, most of the previous molecular biomarkers were unable to cope with batch effects and rely on normalization algorithms, which might result in “test set bias”. This study aims to develop and validate a platform-independent single-sample prognostic model for colorectal cancer without the need for normalization. Methods: A total of 2510 colorectal cancer patients from 13 microarray or sequencing cohorts with complete follow-up data were included in this study. The resampling algorithm and gene pair method were combined to improve the stability of the model. After 1000 times of resampling in the training cohort, an ensemble model was constructed by selecting the k-TSP (k-Top Scoring Pairs) classifiers with high balanced accuracy. The Decision-making process in each level was generated by voting, without the need for any cutoff value. C index and D index were used to evaluate the performance of the model. Results: We constructed an ensemble model called “PIANOS”, which could significantly predict the prognosis of CRC in all 13 cohorts. The meta-estimated C index and D index of the PIANOS model were 0.74 (95%CI: 0.71-0.78, P &amp;lt; 0.001) and 2.77 (95%CI: 2.37-3.18, P &amp;lt; 0.001), respectively. After adjusting for clinicopathological factors, PIANOS was still independently associated with the prognosis of CRC. PIANOS also showed superior prognostic value when compared with previously published gene signatures or consensus molecular subtypes (CMS). GSEA analysis found that the high-risk group was enriched in angiogenesis, TGF-β and epithelial-mesenchymal transition (EMT) pathway, which was consistent with the characteristics of the CMS4 subtype in colorectal cancer. Conclusion: The PIANOS could robustly predict the prognosis of colorectal cancer across different cohorts and platforms, without the need for any normalization, which is promising for refining treatment decisions in clinical practice. Citation Format: Du Cai, Haoning Qi, Wei Wang, Min-Er Zhong, Dejun Fan, Cheng-Hang Li, Xiaojian Wu, Feng Gao. PIANOS: A platform independent and normalization free single-sample classifier for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5126.
Abstract 3205: Role of RIG-I in tumor endothelium
Authors: Adrian M. Baris, Eugenia Fraile-Bethencourt, Sokchea Khou, Rebecca Ruhl, Clay Hudson, Sudarshan Anand
Journal: Cancer Research (2022)

Abstract The immune microenvironment plays a critical role in the efficacy of cancer treatment. The manipulation of cytosolic nucleic acid sensors has become a strategy to boost the immune response in cancer therapy, but it is critical to understand how these nucleic acid sensors work in the tumor microenvironment. One aspect of the immune microenvironment is the nucleic acid sensor, which recognizes pathogenic nucleic acids and activates an immune response. Retinoic acid-inducible gene I (RIG-I) is an RNA sensor that responds to dsRNA by activating a type I interferon response. It has been found that RIG-I activation can induce an anti-tumor immune response, but its role in the tumor microenvironment is unknown. Prior studies show regulation of the innate immune response in endothelium can increase the anti-tumor response. Understanding the role of RIG-I in endothelial cells, and how this can be used to potentially reprogram the tumor microenvironment, is critical for developing more effective cancer treatments. In addition, my research on the effects of RIG-I in endothelium has wide-reaching implications beyond cancer treatment. I hypothesize RIG-I activation in endothelial cells will produce an anti-tumor immune response. My findings show that endothelial RIG-I activates a robust type I interferon response and inhibits EC function including, inducing apoptosis and inhibiting migration, which is indicative that RIG-I plays a role in the endothelial immune response. RIG-I also plays a role in the expression of the several angiogenic regulators in a tissue-specific manner. This research will provide critical knowledge about the role of RIG-I in tumor angiogenesis, and provide novel treatment avenues for solid tumors. Citation Format: Adrian M. Baris, Eugenia Fraile-Bethencourt, Sokchea Khou, Rebecca Ruhl, Clay Hudson, Sudarshan Anand. Role of RIG-I in tumor endothelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3205.
Abstract 2822: Enhanced effector activity of mediator CDK8 kinase module deficient CAR-T Cells
Authors: Katherine A. Freitas, Julia A. Belk, Elena Sotillo, Bence Daniel, Katalin Sandor, Dorota Klysz, Vandon T. Duong, Peng Xu, Meena Malipatlolla, Evan W. Weber, Robbie G. Majzner, Howard Y. Chang, Ansuman T. Satpathy, Crystal Mackall
Journal: Cancer Research (2022)

Abstract Adoptive T cell immune therapies mediate impressive clinical benefit in a fraction of patients, but anti-tumor effects are often limited by inadequate T cell potency. To identify genes that limit T cell effector function, we conducted genome-wide CRISPR knock-out screens in human primary CAR-T cells. The top hits were components of the CDK8 kinase module of the Mediator complex, an evolutionarily conserved regulator of gene transcription. CDK8 kinase module deficient CAR-T cells manifest increased expansion, cytokine production, metabolic fitness, effector function, anti-tumor activity and reduced terminal effector differentiation. CDK8 kinase module deficient CAR-T cells showed widespread but selective increases in chromatin accessibility, MED1 chromatin occupancy, and H3K27 acetylation most notably involving transcription factors that play a critical role in T cell fate, including several STAT and AP1 family members. The most pronounced enhancement was observed for STAT5 which manifested as increased sensitivity to IL-2 in CDK8 kinase module deficient CAR-T cells. These results link Mediator induced transcriptional coactivation with T cell effector programming and identify the CDK8 kinase module as a target for enhancing the potency of anti-tumor T cell responses. Citation Format: Katherine A. Freitas, Julia A. Belk, Elena Sotillo, Bence Daniel, Katalin Sandor, Dorota Klysz, Vandon T. Duong, Peng Xu, Meena Malipatlolla, Evan W. Weber, Robbie G. Majzner, Howard Y. Chang, Ansuman T. Satpathy, Crystal Mackall. Enhanced effector activity of mediator CDK8 kinase module deficient CAR-T Cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2822.
Abstract 3887: β3-adrenergic receptor sustains IFN-γ-dependent PD-L1 expression and impairs anti-tumor immunity in neuroblastoma
Authors: Gennaro Bruno, Nicoletta Nastasi, Angela Subbiani, Alessia Boaretto, Annalisa Tondo, Claudio Favre, Maura Calvani
Journal: Cancer Research (2022)

Abstract In this study we aimed to investigate the role of the β3-adrenergic receptor (β3-AR) in regulating the immune system response against neuroblastoma (NB) tumor. NB is a very heterogeneous extracranial tumor occurring in childhood. A distinctive feature of NB tumors is their neuroendocrine ability to secrete catecholamines, which in turn, via β-adrenergic receptors ligation, may affect different signaling pathways in the tumor microenvironment (TME). We previously demonstrated that the specific antagonism of the β3-AR on NB tumor cells affected tumor growth and progression. Here, in a murine syngeneic model of NB (A/J mice inoculated with the murine neuroblastoma cell line Neuro-2a), the β3-AR blockade ability to influence the number of immunoreactive and/or immunosuppressive cells in TME was investigated through flow cytometry analysis. Moreover, the involvement of the immune-checkpoint signaling axis PD-1/PD-L1 in mediating the anti-tumoral effects brought by a β3-AR antagonist administration was analyzed. Results demonstrated that β3-AR antagonism leads to an immune response reactivation, partially dependent on the PD-1/PD-L1 signaling axis involvement. Indeed, β3-AR antagonism was able to increase the number of immune reactive CD8+, natural killer (NK) and dendritic cells (DCs), and to decrease the number of immune suppressive regulatory T cells (Treg) and Myeloid-derived suppressor cells (MDSC) in tumor mass. Moreover, β3-AR blockade on tumor infiltrating lymphocytes (TILs) dampened their ability to secrete IFN-γ, which in turn reduced the PD-L1 expression on NB tumor cells. Further investigations, through a genomic analysis on NB patients, showed that high ADRB3 gene expression correlates negatively with the patient’s clinical outcome compared to the low expression group, and that ADRB3 gene expression is also related to the expression of different immune-checkpoints. Overall, results indicate that β3-AR in NB TME is able to modulate the interaction between tumor and host immune system, and that its antagonism hits multiple pro-tumoral signaling pathways. Citation Format: Gennaro Bruno, Nicoletta Nastasi, Angela Subbiani, Alessia Boaretto, Annalisa Tondo, Claudio Favre, Maura Calvani. β3-adrenergic receptor sustains IFN-γ-dependent PD-L1 expression and impairs anti-tumor immunity in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3887.
Abstract 2403: Regulation of FAK phosphorylation by serine/threonine protein phosphatases in cancer cells
Authors: Lin Li, Lie Liu, Shile Huang
Journal: Cancer Research (2022)

Abstract Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, is activated by growth factors and integrin clustering, promoting cell migration, a critical step for cancer metastasis. The activity of FAK is closely associated with the phosphorylation of diverse residues, which are mediated by various protein tyrosine kinases and phosphatases. Previous studies have demonstrated that serine/threonine protein phosphatase 5 (PP5) promotes cell migration, while protein phosphatase 2A (PP2A) inhibits cell migration. However, whether PP5 and PP2A regulate cell migration by mediating FAK phosphorylation is poorly understood. In this study, we found that overexpression of PP5 decreased FAK phosphorylation at S722, and knockdown of PP5 increased p-FAK (S722) in a spectrum of tumor cell lines. Our co-immunoprecipitation (co-IP) results showed an interaction between PP5 and FAK. In addition, we observed that overexpression of PP5 induced S178 phosphorylation of paxillin, an FAK interacting protein. However, there was no physical interaction between PP5 and paxillin, indicating an indirect regulation. In addition, knockdown of PP2A enhanced the phosphorylation of FAK and conferred resistance to rapamycin inhibition of IGF-I-stimulated phosphorylation of FAK. To our surprise, by co-IP, we failed to detect any direct interaction between FAK and PP2A. Further research will determine how PP5 and PP2A regulate cell migration through FAK and paxillin. Acknowledgements This work was supported by the Feist-Weiller Cancer Center (FWCC) Bridge Award (S.H.), Shreveport, LA, USA Citation Format: Lin Li, Lie Liu, Shile Huang. Regulation of FAK phosphorylation by serine/threonine protein phosphatases in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2403.
Abstract 6070: Functional natural killer cells derived from engineered hiPSC with hypoimmunity gene combo demonstrate hypoimmunity features in evading host attacks
Authors: Jiabiao Hu, Xiangjun He, Jing Xu, Yixuan Zhou, Yanan Yue, Yangbin Gao, George Church, Luhan Yang
Journal: Cancer Research (2022)

Abstract Engineered human induced pluripotent stem cells (hiPSC)-derived allogeneic Natural Killer (NK) cells are emerging as promising safe and effective off-the-shelf cell therapy drugs. Repeated dosing of NK cells can partly compensate the relatively shorter persistence of NK cells compared with chimeric antigen receptor T cells (CAR-T); However, with each repeat, the persistence and efficacy are increasingly challenged by activation of host innate and adaptive immunity. Thus, reducing immunogenicity of allogeneic NK cells holds promise for better efficacy. In this study, we report a combination of “hypoimmunity” gene edits including multiple transgene knock-ins and both class I and class II MHC knockouts (2KO). We show that hiPSC engineered by the hypoimmunity combo can be efficiently differentiated into NK cells, comparable to the non-engineered ones. This is contrary to the notion from mouse studies that NK cells devoid of class I molecules are hypo-responsive, these engineered NK cells can be expanded by aAPC (artifitial Antigen Presenting Cells, a K562 cell line with surface expression of mbIL-21 and CD137L) and are capable of killing tumor cells, Additionally, we show that the engineered NK cells can evade T cell immunity, as examined by in vitro T cell proliferation and cytotoxicity assays. A humanized mouse model with transplanted CD34-positive human hematopoietic stem cells confirmed that the engineered NK cells can persist longer than WT and 2KO NK cells. Hypoimmune-QN-019 NK cells are generated by incorporating hypoimmunity gene combo edits into our hiPSC-derived NK cell clinical candidate QN-019 which has 3 transgenes including membrane-bound IL-15, high affinity non-cleavable CD16 and CAR targeting CD19. Comparing to QN-019, hypoimmune-QN-019 displayed better efficacy in humanized CDX mouse model in clearing CD19-positive cancer cells. Taken together, we have engineered hiPSC-derived allogeneic NK cells with hypoimmunity features, which hold great promise in becoming an off-the-shelf drug with improved in vivo persistence and efficacy for repeated dosing. Citation Format: Jiabiao Hu, Xiangjun He, Jing Xu, Yixuan Zhou, Yanan Yue, Yangbin Gao, George Church, Luhan Yang. Functional natural killer cells derived from engineered hiPSC with hypoimmunity gene combo demonstrate hypoimmunity features in evading host attacks [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6070.